Human APOBEC3 exerts a strong selective pressure on HIV-1_JR-CSF containing a frameshift in <i>vif</i>.

<p>(A) Plasma viral load analysis in humanized mice intravenously infected with 9×10⁴ or 3.6×10⁵ TCIU of HIV_JR-CSF<i>vif</i>FS. Viral RNA was not detected in the plasma (circles, n = 10) unless the <i>vif</i> ORF is restored (triangles, n = 6). The appearance of plasma viremia was delayed by 4 weeks in one of these mice. (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice intravenously infected with HIV_JR-CSF<i>vif</i>FS. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Viral DNA is sparsely present in tissues from mice where <i>vif</i> was not restored (indicated as FS1–10). In contrast, all tissues analyzed from the six mice where <i>vif</i> was restored had viral DNA present (FS11–16). N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites (<u>G</u>G, <u>G</u>A, <u>G</u>Y) that were mutated in 76 viral DNA sequences amplified from the tissues of HIV_JR-CSF<i>vif</i>FS infected mice where <i>vif</i> was not restored (40% of all <u>G</u>G sites mutated) or mice where <i>vif</i> was restored (no hypermutation). Data represent mean +/− SEM. (D) G to A mutational profile of all viral DNA from mice infected with HIV_JR-CSFΔ<i>vif</i>. Percentages indicate the proportion of G to A mutations occurring at <u>G</u>G (blue), <u>G</u>A (red), or <u>G</u>Y (black) sites. FS1–FS9, FS11–FS14, NSG-hu mice. FS10, FS15–FS16 NSG-BLT mice.</p

Human APOBEC3 rapidly restricts <i>vif</i>-deleted HIV-1_JR-CSF<i>in vivo</i>.

<p>(A) Replication of HIV_JR-CSF, HIV_JR-CSFΔ<i>vif</i>, and HIV_JR-CSF<i>vif</i>FS in CEM-SS cells expressing CCR5 (CEM-SS CCR5). Culture supernatant was assayed for p24^Gag by ELISA at three day intervals to determine the replication kinetics of the mutant viruses. (B) Nested PCR amplification of viral DNA from the tissues obtained one week post-exposure from a representative NSG-hu mouse infected with 9×10⁴ TCIU of wild-type HIV-1_JR-CSF (WT1) or from three mice infected with 3.6×10⁵ TCIU of HIV_JR-CSFΔ<i>vif</i> (indicated as D1–3). (C) Highlighter sequence analysis of 7 wild-type and 3 Δ<i>vif</i> HIV DNA sequences. Amplified viral DNA from panel A showed no APOBEC3 induced mutations in HIV_JR-CSF (WT1 all sequence from tissues is shown together). In contrast, viral DNA from all positive tissues obtained from HIV_JR-CSFΔ<i>vif</i> infected mice had G to A (green lines) and/or C to T mutations (red lines). HIV-1_JR-CSF nucleotide numbers are indicated at the bottom.</p

Restoration of <i>vif</i> occurs only following direct virus injection into the thymus.

<p>(A) Longitudinal analysis of plasma viral load in NSG-BLT humanized mice infected with HIV_JR-CSF<i>vif</i>FS directly into the human thymic implant, spleen, liver, or lung. Solid symbols represent mice infected with a low dose of virus (9×10⁴ TCIU), open symbols represent the high dose infection (3.6×10⁵ TCIU). (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. (C) Percentage of putative APOBEC3 mutation sites (<u>G</u>G, <u>G</u>A, <u>G</u>Y) that were mutated in 44 viral DNA sequences amplified from the tissues of mice injected with HIV_JR-CSF<i>vif</i>FS. Mice injected into the thymic implant had no <u>G</u>G to <u>A</u>G mutations in any tissue whereas HIV DNA from four tissues of mouse FS24 injected into the spleen is hypermutated. (D) G to A mutational profile of all viral DNA from mouse FS24 which failed to restore the <i>vif</i> ORF. Percentages indicate the proportion of G to A mutations occurring at <u>G</u>G (blue), <u>G</u>A (red), or <u>G</u>Y (black) sites.</p

<i>vif</i>-deleted HIV-1_JR-CSF does not overcome APOBEC3 restriction <i>in vivo</i>.

<p>(A) Longitudinal analysis of plasma viral load in humanized mice intravenously infected with 9×10⁴ TCIU of wild-type HIV-1_JR-CSF or 3.6×10⁵ TCIU of HIV_JR-CSFΔ<i>vif</i>. (B) Detection of HIV DNA (+) by nested PCR from the tissues of humanized mice in panel A. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. N/A = not analyzed. (C) Percentage of putative APOBEC3 mutation sites (<u>G</u>G, <u>G</u>A, <u>G</u>Y) that were mutated in 17 viral DNA sequences amplified from the tissues of infected mice. Viral DNA from mice infected with HIV_JR-CSFΔ<i>vif</i> had 25%–65% of <u>G</u>G sites mutated. (D) G to A mutational profile of all viral DNA from mice infected with HIV_JR-CSFΔ<i>vif</i>. Percentages indicate the proportion of G to A mutations occurring at <u>G</u>G (blue), <u>G</u>A (red), or <u>G</u>Y (black) sites. D4-D8, WT2-WT3, NSG-hu mice. D9-D11, NSG-BLT mice.</p

Model depicting sustained <i>in vivo</i> replication of HIV_LAIΔ<i>vif</i>.

<p>Ongoing viral replication persists in cells with low APOBEC3 (shown in green) with little to no APOBEC3 mutation but infection of cells expressing high APOBEC3 (shown in blue) leads to hypermutation of the viral genome, resulting in lethal restriction of HIV. Replication competent viruses are shown in red, defective virions are shown in black.</p

APOBEC3G expression is reduced in the thymus.

<p>(A) Human A3G mRNA levels from human PBMC (n = 7), human thymus (n = 6), NSG-BLT humanized mouse thymus (n = 18), and CD4⁺ cells from NSG-BLT humanized mouse lung (n = 6), spleen (n = 7), liver (n = 7) were determined using qRT-PCR and normalized to human TATA Box binding protein. Hatched bars represent human cells; open bars represent humanized mouse cells. NS =  not significant, *** p<0.01 by one way ANOVA with Bonferroni's posttest. Data represent mean +/− SEM. (B) Immunoblot for human A3G and GAPDH from human PBMC and cells from human and humanized mouse thymus. Numbers represent A3G normalized to GAPDH compared to human PBMC which is set to 1.</p

Sustained Vif-independent replication of CXCR4 tropic HIV-1.

<p>(A) Plasma viral load was monitored in NSG-BLT humanized mice infected with 3.6×10⁵ TCIU HIV_LAIΔ<i>vif</i> directly into the human thymic implant, spleen, liver, or lung. Direct injection of HIV_LAIΔ<i>vif</i> into the thymus resulted in plasma viremia in 4/4 infections. (B) Longitudinal analysis of plasma viral load in humanized mice infected intravenously with 3.6×10⁵ TCIU of HIV_LAIΔ<i>vif</i> (n = 7) or 3–9×10⁴ TCIU of wild-type HIV_LAI (n = 6). Data represent mean +/− SEM. (C) Longitudinal analysis of the percentage of CD4⁺ T cells in the peripheral blood of humanized mice infected in panel B. (D) Comparison of the G to A mutation frequency in the viral RNA from the plasma and the viral DNA from peripheral blood cells from mice intravenously infected with HIV_LAIΔ<i>vif</i>. Data represent mean +/− SEM from 18 sequences, ** p = 0.0066. (E) Detection of HIV DNA (+) by nested PCR from the tissues of BLT humanized mice in panels A and B. Negative tissues (−) yielded no amplified viral DNA using two independent nested PCR primer sets targeting separate regions of the viral genome. Direct injection of HIV_LAIΔ<i>vif</i> into the liver, lung or spleen resulted in limited tissue distribution of viral DNA. (F) Comparison of the G to A mutation frequency in viral DNA from the thymus compared to viral DNA from other tissues of mice infected intrathymically (n = 4) and intravenously (n = 7) with HIV_LAIΔ<i>vif</i>. Data represent mean +/− SEM from 83 sequences. (G) G to A mutational profile of all viral DNA from mice infected with HIV_LAIΔ<i>vif</i>. Percentages indicate the proportion of G to A mutations occurring at <u>G</u>G (blue), <u>G</u>A (red), or <u>G</u>Y (black) sites.</p

BLT mouse peripheral blood harbors few unswitched and switched memory B cells relative to adult human peripheral blood.

<p>(<b>A–B</b>) Representative flow cytometry analyses for CD27 and IgD expression on B cells in adult human PB and BLT mouse PB shows a paucity of unswitched/switched memory B cells in BLT mouse blood. (adult PB, n = 5; BLT PB, n = 18). (<b>C–D</b>) Flow cytometry plots and bar graph for CD38 and IgD expression on CD19⁺ cells reveal an absence of switched memory B cell populations in BLT mouse PB while these populations are present in adult human PB (adult PB, n = 5; BLT PB n = 16). * indicates a p value less than 0.05. ** indicates a p value less than 0.01. *** indicates a p value less than 0.001. Comprehensive statistical analyses are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108663#pone-0108663-t001" target="_blank">Table 1</a>.</p

PC-KLH induced systemic production of PC-specific IgM expressing plasma cells in BLT mice.

<p>ELISPOT analyses revealed the presence of PC-specific IgM producing plasma cells in the BM (n = 6), spleen (n = 6), LN (n = 6), liver (n = 6) and lungs (n = 3) of immunized BLT mice. PC-specific IgG, KLH-specific IgM and KLH-specific IgG producing plasma cells were rare in each of the tissues sampled. Statistical analyses were 1 way ANOVA with Bonferroni's multiple comparisons tests. *** indicates a p value less than 0.001.</p

B cell populations in BLT mice according to CD27/IgD and CD38/IgD co-expression patterns.

<p>Data presented as Mean ± SD.</p><p>n.s.  =  not significantly different.</p><p>Immature B/Atypical switched memory B =  CD27^neg IgD^neg.</p><p>Naïve Mature B =  CD27^neg IgD⁺.</p><p>Transitional B/Mature Naïve B =  CD27⁺ IgD⁺.</p><p>Switched Memory IgM + B/Plasmablasts  =  CD27⁺ IgD^neg.</p><p>B cell populations in BLT mice according to CD27/IgD and CD38/IgD co-expression patterns.</p